Compounds useful as antiproliferative agents

ABSTRACT

Melatonin compounds are disclosed of the formula ##STR1## where R is hydrogen or C 1  to C 6  linear or branched alkylene substituted with phenyl; R 1  is hydrogen, substituted benzyl, naphthylmethyl or taken together with R 2  and the two carbon atoms of the five-membered hetero ring form the group ##STR2## where R 1  &#39; is C 1  to C 6  linear or branched alkanoyl and R 1  &#34; is hydrogen, C 1  to C 6  linear or branched alkyl or phenyl optionally substituted with one or more halogen, amino, nitro, hydroxy, alkyl, alkoxy or haloalkyl; R 2  is hydrogen, 1-pyrrolyl, 1-pyrrolyl substituted with one or more alkyl or alkoxy, the group --(CH 2 ) m  --NHR 2  &#39;1, where m is 1 to 3 and R 2  &#39; is phenyl sulfonyl, the phenyl group optionally substituted with alkyl, or --C(O)--R 2  &#34;, where R 2  &#34; is C 1  to C 6  linear or branched alkylene substituted with a substituent selected from the group consisting of hydrogen, phenyl, alkoxy, alkoxy substituted with phenyl, carboxylic acid or the alkyl esters thereof, carbamoyloxy alkyl, substituted carbamoyloxy alkyl and amino or 5-(2-alkanoyl) tetrazole; R 3 , R 4 , R 5  and R 6  are the same or different and are C 1  to C 6  linear or branched alkyl, C 1  to C 6  linear or branched alkoxy, phenoxy or phenoxy substituted with one or more C 1  to C 6  linear or branched alkyl. 
     The compounds display melatonin antagonist and are antiproliferative agents.

FIELD OF THE INVENTION

This invention relates to novel 2-aryl substituted tryptamines,carbolines and pyrroles having pharmacological activity, e.g., melatoninantagonist activity and methods for the synthesis thereof.

BACKGROUND OF THE INVENTION

Melatonin, 5-methoxy-N-actyltryptamine, is a major hormone of the pinealgland. The synthesis and secretion of melatonin exhibit a circadianrhythm that changes with the seasons and with age, e.g., pubescence andsenescence. The rhythm appears to be the result of both endogenousmechanisms and environmental cues, most notably, the exposure of mammalsto light, which inhibits melatonin synthesis and secretion. Melatonin isthought to be the hormonal mediator of photoperiodic changes. Evidencesuggests that melatonin is involved in the regulation of circadianrhythms and a variety of neural and endocrine functions. Melatonin hasbeen implicated in a number of human disorders, particularly thoserelating to chronobiologic abnormalities. Researchers have suggestedadministering melatonin to alleviate or prevent disturbances incircadian rhythms caused by the rapid crossing of time zones, e.g., jetlag, or changes in work shifts from night to day. See European PatentApplication 0 126 630, by Short et al.

Melatonin analogues have been studied for their effects on thereproductive system, specifically antigonadal activity and inhibition ofrelease of luteinizing hormone (LH); see Flaugh et al., JournaI ofMedicinaI Chemistry, (1979) 22:63-69.

Frohn et al., Life Sciences, Vol. 27, pp. 2043-2046, Pergamon Press, andClemens et al., J. Neural Transm., (1986) [suppl.] 21:451-459, disclosemelatonin analogues wherein the activity of such analogues is related tostructure. Analogues with improved metabolic stability, e.g.,2-methyl-6,7-dichloromelatonin, are disclosed.

Heward and Hadley, (1975), Life Sci., 17:1167-1168, reported in-vitromethod for measuring the melatonin response of various indoleamines,including melatonin and 5-methoxy-N-acetyltryptamine, by frog skinlightening. Of the compounds disclosed in this article, onlyN-acetyltryptamine and N-acetylserotonin were reported to have"melatonin blocking activity".

Dubocovich, Eur. J. Pharmacol., 105:193-194, 1984, also reported thatN-acetyltryptamine competitively antagonized the inhibitory effect ofmelatonin in the chicken retina, thus demonstrating melatonin blockingactivity by a more sensitive in-vitro method than utilized by Heward andHadley.

In Cattanoch, et al., J. Chem Soc. c(10) 1235-43 (1968) a series oftetrahydro-1H-pyrido [4,3-b] indoles were prepared and examined fortheir ability to block the pharmacological actions of seratonin. The2-acetyl derivatives of 8-chloro-2,3,4,5-tetrahydro-1H-pyrido [4,3-b]indole was synthesized but never tested for any type of physiologicalactivity. Also see ibid c(2) 359-66, (1971) for relatedtetrahydroxyamide [3,4-a] compounds.

Several studies have suggested that the pineal gland may influence thedevelopment and growth of mammary tumors in addition tohormone-responsive reproductive tissue. For example, melatoninadministration has been shown to inhibit, while pineolectomy generallystimulates mammary carcinogenesis. Melatonin has, in fact, been studiedwith an aim to prove that it retards hormone responsive breast cancergrowth by directly inhibiting all proliferation. See Hill, et al.,Cancer Research 40 6121-26, (1988).

It is of continuing interest to identify compounds which can inhibitabnormal cell proliferation, while minimizing side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-6 summarize effects of compounds on cell growth described inExamples 4 and 7, 5 and 3, 2 and 9, 6, 1, as well as 10 and 11.

FIGS. 7-15 describe effects of compounds on cell growth described inExamples 4, 7 and 3, 2, 8, 1, 5, 11, 10, as well as 9.

DESCRIPTION OF THE PREFERRED EMBODIMENT

In the present specification and claims, the following terms apply:

C₁ to C₆ linear or branched alkyl means straight or branched chain alkylgroups having from 1 to 6 carbon atoms and includes for example, methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, secondary butyl, tertiarybutyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 2-methylpentyl and thelike. The term "alkyl" is sometimes used herein to mean C₁ to C₆ linearor branched alkyl.

Optionally substituted phenyl (or phenyl optionally substituted) meansphenyl or phenyl substituted by at least one substituent selected fromthe group consisting of halogen (or halo), i.e., chloro, bromo, fluoroor iodo, amino, nitro, hydroxy, alkyl, alkoxy which means linear orbranched chain alkoxy having 1 to 6 carbon atoms and includes forexample, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy,secondary butoxy, tertiary butoxy, pentyloxy, isopentyloxy and hexyloxy,haloalkyl which means linear or branched chain alkyl (as defined above)substituted with at least one halogen and includes for example,chloromethyl, chloromethyl, fluoromethyl, 3-fluoropropyl, 4-chlorobutyl,dichloromethyl, difluoromethyl, 2, 2-difluoroethyl, 4₁ 4-dichlorobutyl,trichloromethyl, trifluoromethyl, 2, 2, 2-trifluoroethyl, 2, 3,3-trifluoropropyl, 1, 1, 2, 2-tetrafluoroethyl, 2, 2, 3,3-tetrafluoropropyl and the like. In the case of mono substituents, thephenyl group can be substituted at position 2-, 3- or 4-. Di, tri, tetraand penta substituents are also included. Such substituents may beattached to the phenyl ring at any appropriately available site.

C₁ to C₆ linear or branched alkylene means branched chain alkylenegroups having 1 to 6 carbon atoms and includes for example, the groupsmethylene, dimethylmethylene, ethylene 2, 2-dimethylpropylene,2-dimethylbutylene and the like.

C₁ to C₆ linear or branched alkanoyl means straight or branched chainalkanoyl group having 1 to 6 carbon atoms and includes, for example,acetyl, propinyl, butyryl, isobutyryl, pivaloyl, valeryl, hexanoxyl andthe like.

Carbamoyloxyalkyl means that the single substituent on the nitrogen atom(attached to alkylene group) of the carbamoyl moiety is selected fromthe group consisting of hydrogen, linear or branched chain alkyl grouphaving 1 to 6 carbon atoms, phenylalkyl which means the alkyl moiety has1 to 6 liner or branched chain carbon atoms and includes for example,benzyl, 2-phenylethyl, 1-phenylethyl, 3-phenylpropyl, 4-phenylbutyl,6-phenylhexyl and the like and substituted phenylalkyl which means theabove-mentioned phenylalkyl substituted by at least one substituentselected from the group consisting of halo, amino, nitro, hydroxy,alkyl, alkoxy and haloalkyl on the phenyl nucleus and includes forexample, N-methyl carbamoyloxymethyl, N-ethyl carbamoyloxymethyl,N-propyl carbamoyloxymethyl, N-butyl carbamoyloxymethyl,1-(N-methylcarbamoyloxy) ethyl and 2-(N-ethylcarbamoyloxy) ethyl.

Substituted carbamoyloxy alkyl means the above described N-substitutedcarbamoyloxy alkyl moieties bearing substituents on the alkyl group (thegroup attached to the oxygen atom of the carbamoyloxy moiety) andincludes phenyl or optionally substituted phenyl and such as the groups(N-methyl carbamoyloxy)phenylmethyl, N-ethyl carbamoyloxymethylphenyl,N-propyl carbamoyloxymethylphenyl, N-butyl carbamoyloxymethylphenyl,2-(N-methyl carbamoyloxy)-2-phenylethyl, 2-(N-ethylcarbamoyloxy)-2-phenylethyl, 3-(N-methyl carbamoyloxy)-3-phenylpropyl,4-(N-methyl carbamoyloxy)-4-phenylpropyl and the like.

The compounds of the present invention are those represented by theformula ##STR3## where R is hydrogen or C₁ to C₆ linear or branchedalkylene substituted with phenyl;

R₁ is hydrogen, substituted benzyl, naphthylmethyl or taken togetherwith R₂ and the two carbon atoms of the five-membered hetero ring formthe group ##STR4## where R₁ ' is C₁ to C₆ linear or branched alkanoyland R₁ " is hydrogen, C₁ to C₆ linear or branched alkyl or phenyloptionally substituted with one or more halogen, amino, nitro, hydroxy,alkyl, alkoxy or haloalkyl;

R₂ is hydrogen, 1-pyrrolyl, 1-pyrrolyl substituted with one or morealkyl or alkoxy, the group --(CH₂)_(m) NHR₂ ' where m is 1 to 3 and R₂ 'is phenyl sulfonyl, the phenyl group optionally substituted with alkyl,or --C())--R₂ ", where R₂ " is C₁ to C₆ liner or branched alkylenesubstituted with a substituent selected from the group consisting ofhydrogen, phenyl, alkoxy, alkoxy substituted with phenyl, carboxylicacid or the alkyl esters thereof, carbamoyloxy alkyl, substitutedcarbamoyloxy alkyl and amino or 5-(2-alkanoyl) tetrazole;

R₃, R₄, R₅ and R₆ are the same or different and are hydrogen C₁ to C₆linear or branched alkyl, C₁ to C₆ linear or branched alkoxy, phenoxy orphenoxy substituted with one or more C₁ to C₆ linear or branched alkyl;

Compounds of the formula ##STR5## where R₇ is C₁ to C₆ linear orbranched alkanoyl, R₈ and R₉ are the same or different and are C₁ to C₆linear or branched alkyl, and n is 1 to 3.

In the compounds of formula I, it is preferred that R₁ and R₂ are takentogether with two carbon atoms of the hetero form compounds of theformula ##STR6##

In the preferred compounds of formula IA it is most preferred that R,R₄, R₅ R₆ and R₁ " are hydrogen and R₃ is halo, C₁ to C₆ linear orbranched alkoxy or phenyoxy.

Especially preferred in the compounds of formula IA are those where R₃is bromo, methoxy, phenoxy and R₁ ' is acetyl.

Another preferred group of compounds of formula I are those of theformula where R₁, R₃, R₄, R₅, R₆ are hydrogen and R₂ is 5-(alkanoyl)tetrazole.

Especially preferred for such compounds are those where R is hydrogen orbenzyl and R₂ is 5(2-acetyl).

A further preferred group of compounds of formula I are those where R₂is phenylsulfonyl and R, R₁ R₃, R₄, R₅ and R₆ are hydrogen. The phenylmay be unsubstituted or substituted with one or more C₁ to C₆ linear orbranched alkyl or 1-pyrrolyl which may be unsubstituted or substitutedwith one or more C₁ to C₆ linear or branched alkyl. R₂ can also be thegroup --(CH₂)_(m) --NHR'₂ where m is an integer from 1 to 3 and R' isthe group --C(O)--R₂ " where R₂ " is C₁ to C₆ linear or branchedalkylene substituted with a substituent selected from the groupsconsisting of phenyl or C₁ to C₆ linear or branched alkoxy. The alkoxygroup can be unsubstituted or substituted with phenyl, carbamoyloxyalkyl or amino.

Particularly preferred for the above preferred compounds are those whereR₂ is (4-methyl) phenyl sulfonyl, 1-(2, 5-dimethyl) pyrrolyl or thegroup --(CH₂)_(m) --NHR₂ ' where R₂ ' is --C(O)--R₂ " where R₂ " is C₁to C₆ linear or branched alkylene substituted with phenyl or with C₁ toC₆ linear or branched alkoxy and m is as previously defined. The alkoxygroup can be unsubstituted or substituted with phenyl.

Especially preferred are the compounds where R₂ " is C₁ to C₆ linear orbranched ethyl, 2-(N-benzyl carbamoyloxy) ethyl, 3-(N-methylcarbamoyloxy) propyl, 4-(N-methyl carbamoyloxy) butyl and the like.

In the compounds of formula II, it is preferred that R₇ is acetyl.Particularly preferred of the preferred compounds are those where R₈ andR₉ are the same and are acetyl.

In preparing the compounds of formula IA a substituted tryptamine, e.g.,##STR7## where R, R₃ R₄ R₅ and R₆ are as previously defined is treatedwith an aldehyde of the formula R₁ "CHO to produce the carbolineintermediate. Acylation provides the compounds of formula IA.

In preparing the compounds of formula I where R₂ is the group--(CH₂)_(m) --NHR₂ ', the compounds of formula IA are first hydrogenatedto open the 6-membered, nitrogen containing ring (the tetrahydropyridylring). Acylation provides the desired product. Other compounds offormula I can be prepared by using various substituted tryptamines asstarting materials. These starting compounds are not new and theirpreparation is well known to those skilled in the art.

The compounds of formula II can be readily prepared by the reaction of adiaminoalkane, e.g., ethylenediamine, with a diketoalkane such as acetylacetone. The resulting 2,5-dialkyl pyrrole bearing an alkylamino groupcan then be subjected to acylation to provide the desired compounds offormula II.

Specific preferred compounds which are within the scope of thisinvention include:

1-N-Acetyl-3-(3,5-dimethyl-1H-pyrrolyl)ethylamine

N-Benzyloxyoarbonyltryptamine

2-Benzyl-3-ethylaoetamido-5-methoxyindole

2-Benzyl-N-propionyltryptamine

1-Methyl-2-N-acetyl-6-benzyloxy-1,2,3,4-tetrahydro-β-carboline

1-Methyl-2-N-acetyl-6-methoxy-1,2,3,4-tetrahydro-β-carboline

1-N-propionyl-3-(3,5-dimethyl-1H-pryyolyl)ethylamine

2-Benzyl-N-acetyltryptamine

2-(1-Naphthylmethyl)-N-acetyltryptamine

2-(4-Methylphenyl)methyl-N-acetyltryptamine

1-Propyl-2-N-acetyl-6-methoxy-1,2,3,4-tetrahydro-β-carboline

1-Methyl-2-N-acetyl-1,2,3,4-tetrahydro-β-carboline

1-Methyl-2-N-propionyl-6-methoxy-1,2,3,4-tetrahydro-β-carboline

1-Methyl-2-N-acetyl-6-benzyloxy-1,2,3,4-tetrahydro-β-carboline

2-N-acetyl-1,2,3,4-tetrahydro-β-carboline

1-Phenyl-2-N-acetyl-5-bromo-1,2,3,4-tetrahydro-β-carboline

2-N-Acetyl-5-bromo-1,2,3,4-tetrahydro-β-carboline

2-(4-Methoxyphenyl)methyl-3-(N-propionyl)aminoethylindole

2-(4-Tolylmethyl)-3-ethylacetamidoindole

2-(4-Methoxyphenyl)methyl-3-ethylacetamidoindole

The above compounds may be made as described in the examples below.

A preferred embodiment of the present invention comprises a method oftreating any disorder in which it is therapeutic to mimic or inhibitmelatonin function that inhibits tumorigenesis by administering atherapeutically effective amount of one or more of the compounds of thepresent invention to a patient suffering from such disorder.

Another preferred embodiment of the present invention comprises a methodof inhibiting or reducing the growth of cancerous tumor cells byadministering (e.g., injection, oral, topical) atherapeutically-effective amount of one or more of the compounds of thepresent invention to a patient suffering from such disorder.

Another preferred embodiment of the present invention comprises a methodof treating various cell proliferation disorders related to alteredmelatonin function or influenced by melatonin by administering atherapeutically-effective amount of one or more of the compounds of thepresent invention to a patient suffering from one or more of suchdisorders.

Another preferred embodiment of the present invention comprises a methodof treating or inducing various endocrine-related conditions attributedto altered melatonin function or influenced by melatonin particularlyrelating to mammary carcinogenous and the reproductive maturation andfunction, e.g., idiopathic delayed puberty, premature labor, andantifertility, by administering a therapeutically-effective amount ofone or more of the compounds of the present invention to a patientsuffering from such endocrine related conditions. In addition, it isthought that the melatonin agonist compounds of the invention can beused to treat or prevent glaucoma by lowering the intraocular pressureand to manipulate body weight by administering an effective amount ofone or more of the melatonin agonist compounds herein.

In general, a pharmacologically effective daily dose can be from 0.01mg/kg to 25 mg/kg per day, bearing in mind, of course, that in selectingthe appropriate dosage in any specific case, consideration must be givento the patient's weight, general health, metabolism, age and otherfactors which influence response to the drug. A particularly preferreddose is 1.0 mg/kg per day.

Another embodiment of this invention is the provision of pharmaceuticalcompositions in dosage unit form which comprise from about 2 mg. to 500mg. of a compound of the above formula.

The pharmaceutical composition may be in a form suitable for oral use,for example, as tablets, aqueous or oily suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, or syrups orelixirs.

Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents, and preserving agents in order to provide apharmaceutically elegant and palatable preparation. Tablets contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for manufacture of tablets.

These excipients may be, for example, inert diluents, for examplecalcium carbonate, sodium carbonate, lactose, calcium phosphate, orsodium phosphate; granulating disintegrating agents, for example maizestarch, or alginic acid; binding agent, for example starch, gelatine, oracacia; and lubricating agents, for example magnesium stearate, stearicacids, or talc. The tablets may be uncoated or they may be coated byknown techniques to delay disintegration and adsorption in thegastrointestinal tract. Thereby a sustained action over a longer periodcan be provided.

Formulations for oral use can also be presented as hard gelatinecapsules wherein the active ingredient is mixed with an inert soliddiluent, for example calcium carbonate, calcium phosphate, or kaolin, oras soft gelatine capsules wherein the active ingredient is mixed with anoil medkiu, for example arachis oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active compound in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example of polyoxethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol, for example, polyoxyethylene sorbitol monooleate, orcondensation product of ethylene oxide with partial esters derived fromfatty acids and hexitol anhydrides, for example polyoxyethylene sorbitanmonooleate. The said aqueous suspensions can also contain one or morepreservatives, for example ethyl, n-propyl, or p-hydroxy benzoate, oneor more coloring agents, one or more flavoring agents, and one or moresweetening agents, such as sucrose, saccharin, or sodium or calciumcyclamate.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example, sweetening, flavoring, and coloringagents, can also be present.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, sorbitol or sucrose. Such formulation can also contain ademulcent, a preservative and flavoring and coloring agents. Thepharmaceutical compositions can be in the form of a sterile injectablepreparation, for example, as a sterile injectable aqueous suspension.This suspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents which havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1,3-butane diol.

The pharmaceutical compositions of the present invention also includecompositions for transdermal and/or intranasal administration. Thus, thecompounds of the present invention can be compounded with apenetration-enhancing agent such as 1-n-dodecylazacyclopentan-2-one orthe other penetration-enhancing agents disclosed in U.S. Pat. Nos.3,991,203 and 4,122,170 Which are hereby incorporated by reference todescribe penetration-enhancing agents which can be included in thetransdermal or intranasal compositions of this invention.

The pharmaceutical compositions can be tableted or otherwise formulatedso that for every 100 parts by weight of the composition there arepresent between 5 and 95 parts by weight of the active ingredient. Thedosage unit form will generally contain between about 1 mg. and about100 mg. of the active ingredient of the formula stated above.

From the foregoing formulation discussion it is apparent that thecompositions of this invention can be administered orally orparenterally. The term parenteral as used herein includes subcutaneousinjection, intravenous, intramuscular, or intrasternal injection orinfusion techniques.

To illustrate the manner in which the invention can be carried out, thefollowing examples are given. It is understood, however, that theexamples are for the purpose of illustration and the invention is not tobe regarded as limited to any of the specific materials or conditionstherein.

EXAMPLE 1 PREPARATION OF1-N-ACETYL-3-(3,5-DIMETHYL-1H-PYRROLYL)ETHYLAMINE

To a stirred solution of 0.50 g (.083 mol) of ethylene diamine and 9.49g (.083 mol) of acetonyl acetone in 200 mLs toluene was added five dropsof acetic acid. The mixture was brought to reflux overnight under aDean-Stark trap and after workup yielded the product which showedcharacteristic peaks: NMR (300 MHz, CDCl₃) δ 3.85 (t, 2H), 2.95 (t, 2H),2.25 (s, 6H).

To 1.0 g (7.2 mmol) of the above product and 3.0 mL (0.022 mol)triethylamine in toluene (0° C.) was added 0.57 g (7.2 mmol) acetylchloride and the mixture was stirred at RT for 20 min. After workup andpurification by flash chromatography (Silica, 8:2 EtOAc:pet. ether) theproduct was isolated and showed characteristic peaks: NMR (300 MHz,CDCl₃) δ 3.9 (t, 2H), 3.45 (m, 2H), 2.2 (s, 6H), 2.0 (s 3H).

Anal. calc. for C₁₀ H₁₆ N₂ O: C, 66.64; H, 8.95; N, 15.54. Observed : C,66.82; H, 9.02; N, 15.63.

EXAMPLE 2 PREPARATION OF N-BENZYLOXYCARBONYLTRYPTAMINE

To a mixture of tryptamine 1.0 g (6.2 mmol) in a mixture of CHCl₃ and H₂O, was added 1.1 g (6.2 mmol) benzylchloroformate, followed by dropwiseaddition of sat. NaHCO₃ solution to maintain alkaline conditions. Thereaction was stirred for 2 hrs. The reaction was extracted with CHCl₃and the organic layer was concentrated to dryness giving a dark yellowoil. The oil was crystallized from ethanol/water to give an off-whitecrystalline product.

Anal. calc. for C₁₈ H₁₈ N₂ O₂ : C, 73.0 ; H, 6.1; N, 10.0. Observed : C,73.31; H, 6.05; N, 9.72.

EXAMPLE 3 PREPARATION OF1=METHYL-2-N-ACETYL-6=METHOXY-1,2,3,4-TETRAHYDRO-δ-CARBOLINE

To a suspension of 3.0 g (15.8 mmol) 5-methoxytryptamine in water wasadded concentrated HC₁ dropwise until a solution was obtained, giving apH of 3.0. Then 8.0 mL acetaldehyde was added and the reaction placed inan oil bath at 85° C. and stirred for 1.25 hrs. The reaction was cooledto room temperature, acidified with concentrated HC₁ to pH 1, andconcentrated in vacuo to give an orange brown solid, which was dissolvedin water and made basic with NH₄ OH. After extraction with CH₂ Cl₂, theorganic layer was dried over MgSO₄, filtered and concentrated in vacuoto give an off-white solid, which showed characteristic peaks: NMR(CDCl₃): δ: 1.45 ppm (d, 3H), 3.85 ppm (s, 3H).

To a solution of 700 mg (3.24 mmol)1-methyl-6-methoxy-1,2,3,4-tetrahydro-β-carboline, and.5 drops NO₄ OH inethyl acetate was added 0.4 mL (3.88 mmol) acetic anhydride under N₂gas. The reaction was stirred for 20 min. and then washed with water.The organic layer collected was dried over MgSO₄, filtered, andconcentrated in vacuo to a dark oil. The oil was dissolved in methanoland chromatographed on reverse phase C-18 using methanol and water. Theproduct was collected and concentrated to a white solid, which showedcharacteristic peaks: NMR (CDCl₃) δ: 1.45 ppm (d, 3H), 2.25 ppm (s, 3H),3.85 ppm (s, 3H).

Anal. calc. for C₁₅ H₁₉ N₂ O₂ : C, 69.47; H, 7.38; N, 10.80. Observed:C, 69.21; H, 7.12; N, 10.93.

EXAMPLE 4 PREPARATION OF 2-BENZYL-3-ETHYLACETAMIDO-5-METHOXYINDOLE

To a solution of 3.05 g (0.0288 mol) of benzaldehyde in 250 mL toluenewas added 5.0 g (0.0261 mol) of 5-methoxytryptamine. The solution wasrefluxed for 16 hours with removal of water by a Dean-Stark trap. Themixture was allowed to cool and 3 mL of conc. HC₁ was added and thereaction refluxed for an additional 1 hour until all the water wasremoved. The reaction was then cooled and the product filtered off andwashed with cold EtOAC and cold pet. ether. The product was dissolved inwater and adjusted to pH 10 with conc. NO₄ OH and extracted into CH₂Cl₂. The solvent was removed under reduced pressure to give a yellow oilwhich was purified by flash chromatography on Silica (50:50 EtOAc:petether to 100% EtOAc) giving 3.0 of product (41%) as a ale-yellow solid.

Into a Parr high pressure reactor were placed 3.0 g of the aboveproduct, 15 mL of glacial acetic acid, 90 mL of deionized water, 10 mLof methanol and 0.5 g of 10% Pd/C. The apparatus was assembled andflushed 3× with hydrogen, then placed under 110 psi hydrogen and heatedto 35° C. with stirring for 4 hours. The reaction was filtered and madebasic (pH 10) with conc. NO₄ OH and extracted with CH₂ Cl₂ (3×). Thesolvent was dried (MgSO₄), filtered, and removed under reduced pressureto give 2.76 g of a colorless oil (91%).

Dissolved in 50 mL of EtOAc was 0.25 g of 2-benzyl-5-methoxytryptamine(0.9 mmol) and 3 drops of conc. NO₄ OH. Then 0.25 mL of acetic anhydridewas added and the reaction was stirred for 15 minutes under nitrogen.The reaction was washed with water (3×), washed once with sat. NaClsolution, dried (MgSO₄), filtered, and evaporated under reduced pressureleaving an orange oil, which was purified on HPLC: reverse phase,solvent 55% MeOH→60% MeOH gradient curve, flow=8 mL/min. The compoundwas collected, methanol removed under reduced pressure and the waterremoved, leaving 0.2 g of a pale yellow solid (69%), which showedcharacteristic peaks: NMR (CDCl₃) δ: .1.8 (s 3H), 3.9 (s, 3H), 4.1 (s,2H).

EXAMPLE 5 RATION OF 2-BENZYL-N-PROPIONYLTRYPTAMINE

To 2.3 g (9.2 mmol) of 2-benzyltryptamine in ethyl acetate was added 5drops NO₄ OH and excess K₂ CO₃. The mixture was stirred under N₂ and 1.8g (13.8 mmol) propionic anhydride was added. The reaction was washedwith sat. NaC₁ soln. then H₂ O. The organic layer was dried over MgSO₄,filtered, and concentrated in vacuo to give a brown oil. The oil waspurified by reverse phase chromatography using methanol/water, giving awhite solid showing characteristic peaks: NMR (CDCl₃) δ: 1.0 ppm (t, 3H,2.3 (q, 2H), 4.0 (s, 2H). mp. 100°-201° C.

EXAMPLE 6 PREPARATION OF 2-N-ACETYL-1,2,3,4-TETRAHYDRO-β-CARBOLlNE

To a solution of 3.2 g (20 mmol) tryptamine in 20 ml 1N hydrochloricacid was added 2.2g (24 mmol) glyoxylic acid monohydrate in 70 ml water.This solution was stirred for 1 hour at reflux, cooled, renderedstrongly basic with concentrated ammonium hydroxide, whereupon an oilyresidue appeared. Continuous stirring resulted in solidification of thiscrude product, which was crystallized from ethanol/water. Drying gave asolid with a melting point of 206°-209° C. This product was acetylatedanalogous to the procedure used in Example 3 to give an off-white solidproduct with a melting point of 240°-241° C.

Anal. calc. for C₁₃ H₁₄ N₂ O: C, 72.87; H, 6.59; N,13.07. Observed : C,72 68; H, 6.65; N, 12.95.

EXAMPLE 7 PREPARATION OF1-METHYL-2-N-ACETYL-6-BENZYLOXY-1,2,3,4-TETRAHYDRO-β-CARBOLINE

The procedure of the first step Example 3 was used, substituting5-benzyloxytryptamine for 5-methoxytryptamine. This resultant materialwas acetylated employing the same procedure as in the second step ofexample 3, and the resultant product crystallized from ethyl acetate.The product showed characteristic peaks: NMR (CDCl₃): δ: 1.45 (d, H),5.1 (s, H).

Anal. calc. for C₂₁ H₂₃ N₂ O₂ : C, 75.20; H, 6.91; N, 8.35. Observed :C, 75.32; H, 6.74; N, 8.52.

EXAMPLE 8 PREPARATION OF 2-BENZYL-3-ETHYLACETAMIDOINDOLE

The procedure of Example 4 was used, substituting tryptamine for5-methoxytryptamine. NMR (CDCl₃): δ: 1.7 (s, H), 2.9 (t, 2H), 3.4 (dd,2H), 4.0 (s, 2H), 5.6 (s, 1H), 7.0-7.3 (m, 8H), 7.5 (d, H), 8.4 (s, 1H).

Anal. calc. for C₂₁ H₂₃ N₂ O₂ (0.5 mole H₂ O) : C, 75.72; H, 7.02; N,9.29. Observed : C, 75.58; H, 6.88; N, 9.08.

EXAMPLE 9 PREPARATION OF 2-(1-NAPHTHYLMETHYL)-3-ETHYLACETAMIDOINDOLE

The procedure of Example 4 was used, substituting 1-naphthaldehyde forbenzaldehyde and tryptamine for 5-methoxytryptamine.

EXAMPLE 10

PREPARATION OF 2-(4-TOLYLMETHYL)-3-ETHYLACETAMIDOINDOLE

The procedure of Example 4 was used, substituting p-tolualdehyde forbenzaldehyde and tryptamine for 5-methoxytryptamine.

EXAMPLE 11 PREPARATION OF2-(4-METHOXYPHENYL)METHYL-3-ETHYLACETAMIDOINDOLE

The procedure of Example 4 was used, substituting p-anisaldehyde forbenzaldehyde and tryptamine for 5-methoxytryptamine.

EXAMPLE 12

The above examples were tested for their ability to inhibit the growthof both estrogen positive (MCF-7) and estrogen negative (Hs0578t) humanbreast cancer cell lines in culture. The antiproliferative effects ofthese compounds were compared with melatonin, a reference molecule knownto inhibit growth of estrogen positive cell lines, and, at highconcentrations, can inhibit estrogen negative cell lines such asHs0578t.

MCF-7 cells were maintained in Dulbecco's modified Eagle's medium(DMEM), supplemented with 10% fetal bovine serum (FBS), penicillin(10,000 units/ml) and streptomycin (10 mg/ml) at 37° C in a humidifiedatmosphere containing 5% CO₂. Hs0578t cells were maintained undersimilar conditions, however DMEM containing high glucose was used and 10ug/ml of insulin was added to the medium.

Prior to each experiment, cells from stock flasks were suspended bytreatment with 0.2% EDTA in phosphate buffered saline (pH 7.3). Thecells were then centrifuged at 1000 rpm for 7 minutes, then resuspendedin fresh medium and counted on a hemocytometer. Cells were adjusted to adensity of 3.0×10⁶ /plate in 5 mls of supplemented plating medium. After5 hours, the plating medium was replaced with fresh medium containingeither melatonin or the above examples in concentrations ranging from10⁻¹⁵ M to 10⁻¹⁵ M. These compounds were initially dissolved in ethanoland diluted to the appropriate final concentration with DMEM; the finalconcentration of ethanol per plate was 0.005%. Control cells wereexposed to the ethanol vehicle alone (0.005%). Five days after initialseeding, the cells were resuspended with PBS-EDTA and counted on ahemocytometer to determine the total cell counts per plate.

Each experiment was run using triplicate plates for each concentrationof melatonin or above example as well as the vehicle controls. Cellgrowth in each of the experimental plates was expressed as a percent ofcontrol growth (100%). A given concentration of either an above exampleor of melatonin was considered to be inhibitory if it decreased cellproliferation by 20% or more as compared with controls. Since most ofthe dose-response curves were irregular, the approximate IC₅₀ for eachexample was estimated from a best fit (manually fitted) curve spanningfrom the 100% control growth level to the maximal inhibitoryconcentration of that example.

Effects of the above examples on estrogen receptor positive and estrogenreceptor negative human breast cancer cell lines (MCF-7 and Hs0578t,respectively) are summarized in FIGS. 1-15, and discussed below. FIGS.1-6 summarize effects on the MCF-7 cell line, while FIGS. 7-15 describeeffects on the Hs0578t cell line.

In FIG. 1, it can be seen that Examples #4 and 7 cause an inhibition ofMCF-7 cell growth. Example #4 shows an IC₅₀ of about 6×10⁻¹⁶ M ascompared with an IC₅₀ of 1×10⁻¹⁴ M for melatonin, making this example 60times more potent than melatonin. All concentrations of Example #4 wereinhibitory to cell growth. The maximal inhibitory effect of Example #4was 35% at 10⁻¹³ M Example #7 has an ₅₀ of 1×10⁻¹⁶ M, making this 100times more potent than melatonin. All concentrations of Example #7 wereinhibitory, with the maximal effect of 37% inhibition seen at 10⁻¹⁵ M.

In FIG. 2, Example #3 inhibited MCF-7 cell growth at all concentrations,while Example #5 inhibited cell growth at all concentrations except10⁻¹³ M, 10⁻¹¹ M and 10⁻⁷ M Example #3 has an IC₅₀ of about 8×10⁻¹⁵ M.The IC₅₀ for Example #5 could not be calculated due to the highlyirregular nature of the dose-response curve, however the maximal effectwas seen at 10⁻⁵ M.

FIG. 3 shows inhibition of MCF-7 cell growth at al concentrations forExamples #2 and 9, with the exceptions of Example #2 at 10⁻¹³ M andExample #9 at 10⁻¹³ M and 10⁻⁵ M. Both Example #2 and Example #9 showIC50's of approximately 1×10⁻¹⁶ M.

FIG. 4 shows an IC₅₀ of approximately 1×10⁻¹⁶ M for Example #6. Allconcentrations except 10⁻¹³ M and 10⁻⁵ M were inhibitory to MCF-7 cellgrowth.

FIG. 5 shows that Example #1 all concentrations except 10⁻¹¹ M and 10⁻⁷M inhibited MCF-7 cell growth, with an IC₅₀ of 4×10⁼¹⁶ M.

FIG. 6 shows that Example #10 inhibits MCF-7 cell growth at 10⁻¹⁵M,10⁻¹³ M, and 10⁻¹¹ M, showing an IC₅₀ of 1×10⁻¹⁶ M. Example #11inhibits MCF-7 cell growth at 10⁻¹⁵ M and 10⁻¹³ M, with an IC₅₀ of2×10⁻¹⁶ M.

Continuing with results in the estrogen negative cell line, FIG. 7 showsExample #4 inhibits Hs0578t cell growth at 10⁻¹³ M and 10⁻⁷ M, With anIC₅₀ of 1×10⁻¹⁵ M.

The effects of Examples #7 and 3 on Hs0578t cell growth are summarizedin FIG. 8. All concentrations of these compounds were inhibitory to cellgrowth, with the exception of Example #7 at 10⁻¹¹ M. The IC₅₀ of Example#7 is about 1×10⁻⁸ M, while the IC₅₀ of Example #3 is 1×10⁻¹³ M,

FIG. 9 shows Example #2 inhibits Hs0578t cell growth at 10⁻¹¹ M and 10⁻⁹M, With an IC₅₀ of approximately 8×10⁻¹³ M.

FIG. 10 shows Example #8 inhibits Hs0578t cell growth at 10⁻⁹ M with anIC₅₀ of approximately 1×10⁻¹⁰ M.

Example #1 (FIG. 11) shows inhibition of Hs0578t cell growth only at10⁻⁵ M, while cell growth is actually stimulated at 10⁻⁹ M, resulting inan IC₅₀ of approximately 6×10⁻⁹ M

FIGS. 12-14 show no inhibition of Hs0578t cell growth was observed forExamples #5, 11, and 10.

FIG. 15 shows Example #9 inhibits Hs0578t cell growth at 10⁻⁵ M and 10⁻⁷M, with an IC₅₀ of approximately 4×10⁻⁹ M.

It is clear from the above data that all examples exhibited some degreeof inhibition of the estrogen positive human breast cancer cell lineMCF-7. While the dose-response curves were complex, all the examplestested show IC₅₀ 's in the 10⁻¹⁵ M to 10⁻¹⁶ M range, indicating thatthese analogues are remarkably potent inhibitors of estrogen positivehuman cancer cell growth. In comparison to melatonin, which yielded IC₅₀'s in the 10⁻¹⁴ M to 10⁻¹⁰ M concentration range, the above examples arefrom 60 to one million times more potent inhibitors of MCF-7 cellgrowth.

In the estrogen negative cell line tested (Hs0578t), seven out of tenexamples exhibited some degree of inhibition of cell growth. Melatonin,tested in this cell line, had no inhibitory effect in most experiments.A wide range of IC₅₀ 's was seen (10⁻¹⁵ M to 10⁻⁷ M).

Example #6 appears to be the most potent inhibitor of MCF-7 cell growth,while Example #2 elicits the best maximal inhibitory response. Example#4 appears to be the most potent inhibitor of Hs0578t cells, whileExample #9 shows the best maximal inhibition of growth. Example #11 isnot only a highly potent compound, but it elicits an impressive maximalinhibitory response at a very low concentration. Example #2 is not onlya highly potent inhibitor of both cell lines, but also induces in both asubstantial inhibitory response at low concentrations.

Having now described the invention, I claim:
 1. Compounds of the formula##STR8## where R is hydrogen or C₁ to C₆ linear or branched alkylenesubstituted with phenyl;R₁ is benzyl, or naphthylmethyl; R₂ is the group--(CH₂)_(m) --NHR₂ ', where m is 1 to 3 and R₂ ' is phenyl sulfonyl, thephenyl group optionally substituted with alkyl, or --C(O)--R₂ ", whereR₂ " is C₁ to C₆ linear or branched alkylene substituted with asubstituent selected from the group consisting of hydrogen, phenyl,alkoxy, alkoxy substituted with phenyl, carboxylic acid or the alkylesters thereof, carbamoyloxy alkyl, carbamoyloxy alkyl substituted withphenyl or phenyl substituted with at least one substituent selected fromthe group consisting halo, amino, nitro, hydroxy, alkyl, alkoxy, andhaloalkyl, and amino or 5-(2-alkanoyl) tetrazole; R₃, R₄, R₅ and R₆ arethe same or different and are hydrogen, C₁ to C₆ linear or branchedalkyl, C₁ to C₆ linear or branched alkoxy, phenoxy or phenoxysubstituted with one or more C₁ to C₆ linear or branched alkyl.
 2. Thecompounds according to claim 1 wherein R, R₃ R₄, R₅ and R₆ are hydrogenand R₂ is the group --(CH₂)_(m) --NHR₂ ' where m is 1 to 3 and R₂ ' is--C(O)--R₂ " where R₂ " is C₁ to C₆ linear or branched alkylenesubstituted with a substituent selected from the group consisting ofphenyl, C₁ to C₆ linear or branched alkoxy optionally substituted withphenyl, carboxyl and or the C₁ to C₆ linear or branched alkyl estersthereof, carbamoyloxy alkyl and amino.
 3. The compounds according toclaim 1 wherein R₂ " is C₁ to C₆ linear or branched alkylene substitutedwith C₁ to C₆ linear or branched alkoxy optionally substituted withphenyl.
 4. The compounds according to claim 3 wherein said alkoxy isethoxy.
 5. The compounds according to claim 3 wherein said alkoxy ismethoxy substituted with phenyl.
 6. The compounds according to claim 2wherein R₂ " is C₁ to C₆ linear or branched alkylene substituted withcarboxylic acid and the C₁ to C₆ linear or branched alkyl estersthereof.
 7. The compounds according to claim 2 wherein R₂ " is C₁ to C₆linear or branched alkylene substituted with carbamoyloxy C₁ to C₆ alkyloptionally substituted with phenyl.
 8. The compounds according to claim7 wherein said carbamoyloxy C₁ to C₆ linear or branched alkyl iscarbamoyloxymethyl substituted with phenyl.